This project is designed to answer a number of questions and provide information important for developing a detailed understanding of the beta- oxidation of polyunsaturated fatty acids (PUFA) and hydroxy fatty acids. The focus of this study will be on 2,4-dienoyl-CoA reductase and 3- hydroxyacyl-CoA epimerase which are key enzymes in the reductase-dependent and epimerase-dependent pathways of PUFA beta-oxidation, respectively. Biochemical and genetic approaches will be used to study the beta-oxidation system of mammals and E. coli. An important aspect of this study is an assessment of the importance of 3-hydroxyacyl-CoA epimerase in the beta- oxidation of hydroxy fatty acids like D-5 hydroxymyristic acid in E. coli. Also, the mechanism o the E. coil 3-hydroxyacyl-CoA epimerase will be elucidated as will be in the location of delta3-cis-delta2 trans-enoyl-CoA isomerase on the multienzyme complex of fatty acid oxidation from E. coli. The identification of a human disorder due to a deficiency of 2,4-dienoyl- CoA reductase prompts further studies of this enzyme including (a) the development of a more sensitive assay to measure reductase from rat liver; (c) the cloning, sequencing and overexpression of the E. coli reductase gene; (d) a stereochemical study of the reduction catalyzed by mammalian 2,4-dienoyl-CoA reductase and (e) the hormonal regulation of this enzyme and its effect on PUFA beta-oxidation. Finally, the beta-oxidation of cis and trans unsaturated fatty acids in mitochondria will be studied with the aim of detecting and explaining differences in their degradation. Although, this project will provide a more complete view of the beta- oxidation of polyunsaturated fatty acids and some hydroxy fatty acids in normal an diseased organisms including humans.